EP0549581B1 - Rahmenbau-mutierte antikörper und ihre herstellung - Google Patents

Rahmenbau-mutierte antikörper und ihre herstellung Download PDF

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EP0549581B1
EP0549581B1 EP91907669A EP91907669A EP0549581B1 EP 0549581 B1 EP0549581 B1 EP 0549581B1 EP 91907669 A EP91907669 A EP 91907669A EP 91907669 A EP91907669 A EP 91907669A EP 0549581 B1 EP0549581 B1 EP 0549581B1
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antibody
framework
variable domain
chain
species
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Wellcome Foundation Limited The
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to altered antibodies and their preparation.
  • the invention is typically applicable to the production of humanised antibodies.
  • variable domains of each pair of light and heavy chains form the antigen binding site.
  • the domains on the light and heavy chains have the same general structure and each domain comprises a framework of four regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
  • WO-A-90 07861 relates to a humanised antibody specific for the p55 Tac protein of the IL-2 receptor in which the amino acid sequence of the CDRs is that of a mouse antibody against the same antigen and the amino acid sequence of the variable domain framework regions is that of a human antibody chosen on the basis of homology with the framework regions of the rodent antibody.
  • the DNA encoding the humanised antibody is produced by conventional methods, for example use of synthetic oligonucleotides.
  • the antibody chain may be co-expressed with a complementary antibody chain. At least the framework of the variable domain and the or each constant domain of the complementary chain generally are derived from the said second species also. A light chain and a heavy chain may be co-expressed. Either or both chains may have been prepared by the process of the invention. Preferably the CDRs of both chains are derived from the same selected antibody. An antibody comprising both expressed chains can be recovered.
  • a chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region.
  • the antigen binding region is an antibody light chain variable domain or heavy chain variable domain.
  • the chimaeric antibody comprises both light and heavy chain variable domains.
  • the non-immunoglobulin region is fused at its C-terminus to the antigen binding region.
  • the non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene.
  • the two regions of the chimaeric antibody may be connected via a cleavable linker sequence.
  • the invention is preferably employed to humanise an antibody, typically a monoclonal antibody and, for example, a rat or mouse antibody.
  • the framework and constant domains of the resulting antibody are therefore human framework and constant domains whilst the CDRs of the light and/or heavy chain of the antibody are rat or mouse CDRs.
  • Preferably all CDRs are rat or mouse CDRs.
  • the antibody may be a human IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or IgD carrying rat or mouse CDRs.
  • variable domain framework of the antibody is preferably reshaped to about the closest variable domain framework of an antibody of another species.
  • about the closest is meant about the most homologous in terms of amino acid sequences.
  • Step 1 Determining the nucleotide and predicted amino acid sequence of the antibody light and heavy chain variable domains
  • Step 3 The actual reshaping methodologies/techniques
  • a cDNA encoding the desired reshaped antibody is preferably made beginning with the rodent cDNA from which the rodent antibody variable domain sequence(s) was originally determined.
  • the rodent variable domain amino acid sequence is compared to that of the chosen human antibody variable domain sequence.
  • the residues in the rodent variable domain framework are marked that need to be changed to the corresponding residue in the human to make the rodent framework identical to that of the human framework. There may also be residues that need adding to or deleting from the rodent framework sequence to make it identical to that of the human.
  • Oligonucleotides are synthesised that can be used to mutagenize the rodent variable domain framework to contain the desired residues. Those oligonucleotides can be of any convenient size. One is normally only limited in length by the capabilities of the particular synthesizer one has available. The method of oligonucleotide-directed in vitro mutagenesis is well known.
  • splicing framework regions is technically easier because there is a high degree of homology between the mutagenic oligonucleotide and the rodent variable domain framework. This is true because a human antibody variable domain framework has been selected that is most homologous to that of the rodent.
  • the advantage of the present method of reshaping as opposed to synthesizing the entire reshaped version from scratch is that it is technically easier. Synthesizing a reshaped variable domain from scratch requires several more oligonucleotides, several days more work, and technical difficulties are more likely to arise.
  • Step 4 The transfection and expression of the reshaped antibody
  • the DNA sequence in step a) encodes both the variable domain and the or each constant domain of the antibody chain, the or each constant domain being derived from the first antibody.
  • the antibody can be recovered and purified.
  • the cell line which is transformed to produce the altered antibody may be a Chinese Hamster Ovary (CHO) cell line or an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line.
  • the cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus.
  • the immortalised cell line is a myeloma cell line or a derivative thereof.
  • the cell line used to produce the altered antibody is preferably a mammalian cell line
  • any other suitable cell line such as a bacterial cell line or a yeast cell line
  • E. coli - derived bacterial strains could be used.
  • step (b) may be carried out by further manipulating the vector produced in step (a) so that this vector encodes not only the variable domain of an altered antibody light or heavy chain, but also the complementary variable domain.
  • An antibody is consequently produced in which CDRs of a variable domain of an antibody chain are homologous with the corresponding CDRs of an antibody of a first mammalian species and in which the framework of the variable domain and the constant domains of the antibody are homologous with the corresponding framework and constant domains of an antibody of a second, different, mammalian species.
  • all three CDRs of the variable domain of a light or heavy chain are derived from the first species.
  • the present process has been applied to obtain an antibody against human CD4 antigen. Accordingly, the invention also provides an antibody which is capable of binding to human CD4 antigen, in which the CDRs of the light chain of the antibody have the amino acid sequences:
  • the antibody preferably has the structure of a natural antibody or a fragment thereof.
  • the antibody may therefore comprise a complete antibody, a (Fab') 2 fragment, a Fab fragment, a light chain dimer or a heavy chain.
  • a chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region.
  • the antigen binding region is an antibody light chain variable domain or heavy chain variable domain.
  • the chimaeric antibody comprises both light and heavy chain variable domains.
  • the non-immunoglobulin region is fused at its C-terminus to the antigen binding region.
  • the non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene.
  • the two regions of the chimaeric antibody may be connected via a cleavable linker sequence.
  • the invention is preferably employed to humanise a CD4 antibody such as a rat or mouse CD4 antibody.
  • the framework and the constant domains of the resulting antibody are therefore human framework and constant domains whilst the CDRs of the light and/or heavy chain of the antibody are rat or mouse CDRs.
  • Preferably all CDRs are rat or mouse CDRs.
  • the antibody may be a human IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or IgD carrying rat or mouse CDRs.
  • the framework of the antibody heavy chain is homologous to the corresponding framework of the human antibody KOL (schmidt et al , Hoppe-Seyler's Z. Physiol. Chem., 364 713-747, 1983).
  • the sixth residue of framework 4 in this case is suitably Thr or Pro, preferably Thr.
  • This residue is the 121st residue in the KOL antibody heavy chain variable region (Schmidt et al, 1983), and is identified as residue 108 by Kabat (Kabat et al , "Sequences of proteins of immunological interest", US Dept of Health and Human Services, US Government Printing Office, 1987).
  • the reshaped CD4 antibody can be used to induce tolerance to an antigen. It can be used to alleviate autoimmune diseases such as rheumatoid arthritis. It can be used to prevent graft rejection. Tolerance to a graft such as an organ graft or a bone marrow transplantation can be achieved. Also, the reshaped CD4 antibody might be used to alleviate allergies. Tolerance to allergens could be achieved.
  • a CD4 antibody and, indeed, a CD8 antibody as appropriate are given parenterally, for example intravenously.
  • the antibody may be administered by injection or by infusion.
  • the antibody is formulated in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent. Any appropriate carrier or diluent may be employed, for example phosphate-buffered saline solution.
  • Tolerance can therefore be induced to an antigen in a host by administering non-depleting or depleting CD4 and CD8 mAbs and, under cover of the mAbs, the antigen.
  • a patient may be operated on surgically under cover of the non-depleting or depleting CD4 and CD8 mAbs to be given a tissue transplant such as an organ graft or a bone marrow transplant.
  • tolerance may be induced to an antigen already possessed by a subject. Long term specific tolerance can be induced to a self antigen or antigens in order to treat autoimmune disease such as multiple sclerosis or rheumatoid arthritis. The condition of a patient suffering from autoimmune disease can therefore be alleviated.
  • Figure 1 shows the nucleotide and predicted amino acid sequence of rat CD4 antibody light chain variable region. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively.
  • Base pairs 1-269 HindIII-PvuII
  • 577-620 [Bg1II/Bc1I]-BamHI
  • base pairs 270-576 are from the PCR product of the CD4 antibody light chain variable region (V L ).
  • CDRs boxes
  • CDRs boxes
  • Figure 2 shows the nucleotide and predicted amino acid sequence of the reshaped CAMPATH-1 antibody light chain cDNA. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes.
  • Figure 3 shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody light chain cDNA CD4V L REI. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes.
  • Figure 4 shows the nucleotide and predicted amino acid sequence of rat CD4 antibody heavy chain variable region. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes. Base pairs 1-272 (HindIII-PstI) and 603-817 (BstEII-BamHI) are part of the vector M13V H PCR1, while base pairs 273-602 are from the PCR product of the CD4 antibody heavy chain variable region (V H ).
  • Figure 5 shows the nucleotide and predicted amino acid sequence of the reshaped CAMPATH-1 antibody heavy chain cDNA. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes.
  • Figure 6 shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain cDNA CD4V H NEW-Thr 30 .
  • the number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively.
  • CDRs are identified by boxes.
  • Figure 7 shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain cDNA CD4V H NEW-Ser 30 .
  • the number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively.
  • CDRs are identified by boxes.
  • FIG 8 shows the heavy chain variable (V) region amino acid sequence of the human myeloma protein KOL. CDRs are identified by boxes. This sequence is taken from the Swiss-Prot protein sequence database.
  • Figure 9 shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V H KOL-Pro 113 .
  • the number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively.
  • CDRs are identified by boxes.
  • Figure 10 shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V H KOL-Pro 113 without immunoglobulin promoter. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes.
  • Figure 11 shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V H KOL-Thr 113 .
  • the number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively.
  • CDRs are identified by boxes.
  • Figure 12 shows the nucleotide and predicted amino acid sequence of the reshaped CD4 antibody heavy chain V region CD4V H KOL-Thr 113 without immunoglobulin promoter. The number of the first and last amino acid or nucleotide in each line is indicated in the left and right margins, respectively. CDRs are identified by boxes.
  • the rat-derived anti-human CD4 antibody, clone YNB46.1.8 (IgG 2b , kappa light chain serotype), was the result of fusion between a rat splenocyte and the Lou strain rat myeloma cell line Y3-Ag 1.2.3 (Galfre et al , Nature, 277 : 131-133, 1979) and was selected by its binding to a rat T cell line NB2-6TG stably transfected with an expression vector containing a complementary DNA (cDNA) encoding the human CD4 antigen (Madden et al , Cell, 42 : 93-104, 1985).
  • Antibody was purified by high pressure liquid chromatography (HPLC).
  • Poly(A) + RNA was heated at 70°C for 5 minutes and cooled on ice just prior to use.
  • a 25 ⁇ l first strand synthesis reaction consisted of 5 ⁇ g poly(A) + RNA, 250 ⁇ M each dNTP, 50 mM Tris.HC1 (pH 8.2 at 42°C), 10 mM MgCl 2 , 100 mM KC1, 10 mM dithiothreitol, 23 units reverse transcriptase (Anglian Biotec, Colchester, U.K.), 3.5 pmoles of the V L region-specific oligonucleotide primer V K 1FOR [5'-d(GTT AGA TCT CCA GCT TGG TCC C)] or the V H region-specific primer V H 1FOR-B [5,-d(TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC)], and incubated for 5 minutes at 20°C and then 90 minutes at 42°C.
  • the samples were frozen at -20°C and the mineral oil (a viscous liquid at -20°C) was removed by aspiration.
  • the aqueous phases were thawed, and PCR products were purified by electrophoresis in 2% agarose gels, and then double digested with either PvuII and BglII (V L ) or PstI and BstEII (V H ) restriction enzymes, and cloned into the PvuII and BclI restriction sites of the vector M13V K PCR3 (for V L region; Orlandi et al , 1989) or the PstI and BstEII restriction sites of the vector M13V H PCR1 (for V H region).
  • V L region clones were first screened by hybridisation to a 32 P-labeled oligonucleotide probe [5'-d(GTT TCA TAA TAT TGG AGA CA)] specific for the CDR2 of the Y3-Ag 1.2.3 V L region.
  • V L region clones not hybridising to this probe and V H region clones were sequenced by the dideoxy chain termination method (Sanger et al , PNAS USA 74 : 5463, 1977).
  • the three oligonucleotides [5'-d(AGA GTG ACC ATC ACC TGT CTA GCA AGT GAG GAC ATT TAC AGT GAT TTA GCA TGG TAC CAG CAG AAG CCA), 5'-d(CTG CTG ATC TAC AAT ACA GAT ACC TTG CAA AAT GGT GTG CCA AGC AGA TTC), 5'-d(ATC GCC ACC TAC TAC TGC CAA CAG TAT AAC AAT TAT CCG TGG ACG TTC GGC CAA GGG ACC)] were designed to replace each of the three CDRs in the REI-based human antibody V L region framework that is part of the reshaped CAMPATH-1 antibody V L region (Reichmann et al , 1988).
  • a clone containing each of the three mutant oligonucleotides was identified by nucleotide sequencing and was subcloned into the HindIII site of the expression vector pH ⁇ APr-1 (Gunning et al , PNAS, 84 : 4831-4835, 1987) which also contained a dihydrofolate reductase gene (Ringold et al , J.Mol.Appl. Genet. 1 : 165-175, 1981) driven by a truncated SV40 promoter.
  • CD4V H NEW-Thr 30 was created first by oligonucleotide-directed in vitro mutagenesis in the vector M13mp18 by priming with three oligonucleotides simultaneously on a 1467 base single-stranded cDNA template ( Figure 5) encoding the entire heavy chain of the reshaped CAMPATH-1 antibody (Reichmann et al , 1988).
  • the three oligonucleotides [5'-d(TCT GGC TTC ACC TTC ACC AAC TAT GGC ATG GCC TGG GTG AGA CAG CCA CCT), 5'-d(GGT CTT GAG TGG ATT GGA ACC ATT AGT CAT GAT GGT AGT GAC ACT TAC TTT CGA GAC TCT GTG AAG GGG AGA GTG),5'-d(GTC TAT TAT TGT GCA AGA CAA GGC ACT ATA GCT GGT ATA CGT CAC TGG GGT CAA GGC AGC CTC)] were designed to replace each of the three complementarity determining regions (CDRs) in the NEW-based V H region that is part of the reshaped CAMPATH-1 antibody (Reichmann et al , 1988).
  • CDRs complementarity determining regions
  • FIG. 6 A clone ( Figure 6) containing each of the three mutant oligonucleotides was identified by nucleotide sequencing.
  • CD4V H NEW-Ser 30 was created second by oligonucleotide-directed in vitro mutagenesis in the vector M13mp18 by priming with a single oligonucleotide on the 1458 base single-stranded cDNA template ( Figure 6) encoding CD4V H NEW-Thr 30 .
  • the oligonucleotide [5'-d(GCT TCA CCT TCA GCA ACT ATG GCA T)] was designed to mutate the residue at position 30 from threonine [ACC] to serine [AGC].
  • a clone ( Figure 7) containing this mutant oligonucleotide was identified by nucleotide sequencing. Double-stranded forms of the clones CD4V H NEW-Thr 30 and CD4V H NEW-Ser 30 were subcloned as HindIII fragments into the HindIII site of the expression vector pNH316.
  • the vector pNH316 is a modified version of the vector pH ⁇ APr-1 (Gunning et al , PNAS, 84 : 4831-4835, 1987) which was engineered to contain a neomycin resistance gene driven by a metallothionine promoter.
  • CD4V H KOL-Thr 113 encodes a threonine residue at position 113 ( Figure 11) while the CD4V H KOL-Pro 113 version encodes a proline residue at position 113 ( Figure 9).
  • CD4V H KOL-Thr 113 was created first by oligonucleotide-directed in vitro mutagenesis of single-stranded DNA template containing the 817 base HindIII-BamHI fragment encoding the V H region of the rat CD4 antibody ( Figure 4) cloned into M13mp18 by priming simultaneously with five oligonucleotides [5'-d(CAC TCC CAG GTC CAA CTG GTG GAG TCT GGT GGA GGC GTG GTG CAG CCT GG), 5'-d(AAG GTC CCT GAG ACT CTC CTG TTC CTC CTC TGG ATT CAT CTT CAG TAA CTA TGG CAT G), 5'-d(GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG), 5'-d(ACT ATC TCC AGA GAT AAT AGC AAA AAC ACC CTA TTC CTG CAA ATG G), 5'-d
  • CD4V H KOL-Pro 113 was created second by oligonucleotide-directed in vitro mutagenesis of single-stranded DNA template containing the 817 base HindIII-BamHI fragment encoding CD4V H KOL-Thr 113 cloned into M13mp18 by priming with the oligonucleotide [5'-d(TGG GGC CAA GGG ACC CCC GTC ACC GTC TCC TCA)].
  • a clone containing this mutant oligonucleotide was identified by nucleotide sequencing.
  • the immunoglobulin promoters were removed from the double-stranded DNA forms of clones encoding CD4V H KOL-Thr 113 ( Figure 11) and CD4V H KOL-Pro 113 ( Figure 9) by replacing (for both versions) the first 125 bp (HindIII-NcoI) with a HindIII-NcoI oligonucleotide linker fragment [5'-d(AGC TTT ACA GTT ACT GAG CAC ACA GGA CCT CAC) and its overlapping complement 5'-d(CAT GGT GAG GTC CTG TGT GCT CAG TAA CTG TAA)].
  • the relative affinities of the reshaped antibodies to bind the CD4 antigen were estimated by FACS analysis.
  • the CD4-expressing cells used in this analysis were a cloned rat T cell line NB2-6TG stabily transfected with an expression vector containing a complementary DNA (cDNA) encoding the human CD4 antigen (Maddon et al , Cell, 42 , 93-104, 1985). Cells were stained with the appropriate reshaped antibody followed by fluorescein-conjugated sheep anti-human antibodies (Binding Site Ltd., Birmingham, UK). Control staining (see Table 1) consisted of no antibody present during the first stage of cell staining. Mean cellular fluorescence was determined with an Ortho FACS.
  • the relative avidities of the rat YNB46.1.8 antibody and the reshaped CD4V H KOL-Thr 113 antibody were estimated by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Microtiter plates were coated with soluble recombinant CD4 antigen (Byrn et al , Nature, 344 : 667-670, 1990) at 50 ul/well, 10 ug/ml, and then blocked with 100 ul/well phosphate buffered saline (PBS) containing 1.0% bovine serum albumin (BSA).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • Antibodies were diluted in PBS containing 0.1% BSA, and added to wells (50 ul/well) for 45 minutes at room temperature.
  • Biotinylated CD4V H KOL-Thr 113 antibody (10 ul/well; 20 ug/ml final concentration) was then added to each well for an additional 45 minutes. Wells were washed with PBS containing 0.1% BSA, and then 50 ul streptavidin-biotinylated horseradish peroxidase complex (Amersham; Aylesbury, UK) diluted 1:1,000 was added to each well for 30 minutes. Wells were washed with PBS containing 0.1% BSA, and 100 ul substrate (25 mM citric acid, 50 mM disodium hydrogen phosphate, 0.1% (w/v) o-phenylene diamine, 0.04% (v/v) 30% hydrogen peroxide) was added to each well. Reactions were stopped by the addition of 50 ul/well 1.0 M sulfuric acid. Optical densities at 492 nanometers (OD 492 ) were determined with an ELISA plate reader.
  • Dihydrofolate reductase deficient chinese hamster ovary (CHO DHFR -) cells (10 6 /T-75 flask) were cotransfected as described (Wigler et al , PNAS USA 76 , 1373, 1979) with 9 ⁇ g of heavy chain construct and 1 ⁇ g of the light chain construct. Transfectants were selected in medium containing 5% dialysed foetal bovine serum for 2 to 3 weeks, and antibody-secreting clones were identified by ELISAs of conditioned media. Antibody was concentrated and purified by protein-A Sepharose (Trade Mark) column chromatography.
  • V L and V H regions from CD4 antibody-secreting hybridoma cells were isolated by PCR using primers which amplify the segment of mRNA encoding the N-terminal region through to the J region (Orlandi et al , 1989).
  • V L and V H region PCR products were subcloned into the M13-based vectors M13V K PCR3 and M13V H PCR1, respectively.

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Claims (18)

  1. Verfahren zur Herstellung einer Antikörperkette, in der die Komplementarität-bestimmenden Regionen (CDRs) der variablen Domäne der Antikörperkette von einer ersten Säugerart abgeleitet sind und die Gerüstregion der variablen Domäne und, sofern vorhanden, die oder jede konstante Domäne der Antikörperkette von einer zweiten anderen Säugerart stammen, wobei man
    (i) die die Gerüstregion codierenden Regionen der DNA, die eine variable Domäne einer Antikörperkette der ersten Art codiert, so mutiert, daß die mutierten, die Gerüstregion codierenden Regionen die von der zweiten Art abgeleitete Gerüstregion codieren; und
    (ii) die Antikörperkette exprimiert, wobei die mutierte DNA aus Stufe (i) verwendet wird;
    wobei die Mutation in Stufe (i) so ist, daß ein Antikörper, der die in Stufe (ii) exprimierte Antikörperkette umfaßt, die Bindungsfähigkeit des Antikörpers, von dem die CDRs abgeleitet sind, beibehält.
  2. Verfahren nach Anspruch 1, wobei die die Gerüstregion codierenden Regionen der DNA, die die variable Domäne einer schweren Kette eines Antikörpers codiert, in Stufe (i) mutiert werden.
  3. Verfahren nach Anspruch 1 oder 2, wobei die die Gerüstregion codierenden Regionen der DNA, die die variable Domäne einer leichten Kette eines Antikörpers codiert, in Stufe (i) mutiert werden.
  4. Verfahren nach einem der vorstehenden Ansprüche, wobei die erste Art eine Ratte oder eine Maus ist.
  5. Verfahren nach einem der vorstehenden Ansprüche, wobei die zweite Art der Mensch ist.
  6. Verfahren nach einem der vorstehenden Ansprüche, wobei man
    (a) die Nucleotid- und vorhergesagte Aminosäuresequenz einer variablen Domäne einer ausgewählten Antikörperkette der ersten Art bestimmt;
    (b) die Antikörpergerüstregion bestimmt, gegenüber der die Gerüstregion der Domäne verändert werden soll;
    (c) die die Gerüstregion codierenden Regionen der DNA, die die variable Domäne codiert, so mutiert, daß die mutierten, die Gerüstregion codierenden Regionen die in Stufe (b) bestimmte Gerüstregion codieren;
    (d) die in Stufe (c) erhaltene mutierte DNA an die DNA knüpft, die eine konstante Domäne der zweiten Art codiert, und die DNA in einem Expressionsvektor cloniert; und
    (e) den Expressionsvektor in eine kompatible Wirtszelle einschleust und die Wirtszelle unter solchen Bedingungen züchtet, daß die Antikörperkette exprimiert wird.
  7. Verfahren nach Anspruch 6, wobei die Gerüstregion mit etwa der meisten Homologie einer Antikörperkette einer anderen Art in Stufe (b) als die Gerüstregion ausgewählt wird, gegenüber der die variable Domäne verändert werden soll.
  8. Verfahren nach einem der vorstehenden Ansprüche, wobei der Antikörper der ersten Art ein CD4-Antikörper ist.
  9. Verfahren nach einem der vorstehenden Ansprüche, wobei die Antikörperkette mit einer komplementären Antikörperkette gleichzeitig exprimiert wird und ein Antikörper, der die zwei Ketten umfaßt, isoliert wird.
  10. Antikörper, der an menschliches CD4-Antigen binden kann, wobei die CDRs der leichten Kette des Antikörpers die Aminosäuresequenzen besitzen:
    CDR1:   LASEDIYSDLA
    CDR2:   NTDTLQN
    CDR3:   QQYNNYPWT
    und wobei die CDRs der schweren Kette des Antikörpers die Aminosäuresequenzen besitzen:
    CDR1:   NYGMA
    CDR2:   TISHDGSDTYFRDSVKG
    CDR3:   QGTIAGIRH
    und wobei die Gerüstregion der variablen Domäne und, sofern vorhanden, die oder jede konstante Domäne jeder Kette von einer Nicht-Ratten-Säugerart abgeleitet sind.
  11. Antikörper nach Anspruch 10, wobei die Nicht-Ratten-Säugerart der Mensch ist.
  12. Antikörper nach Anspruch 11, wobei die Gerüstregion der variablen Domäne der schweren Kette der Gerüstregion der variablen Domäne der schweren Kette des Proteins KOL homolog ist.
  13. Antikörper nach Anspruch 12, wobei die variable Region der schweren Kette die Aminosäuresequenz, die in der obersten Zeile der Fig. 10 oder 12 gezeigt ist, besitzt.
  14. Antikörper nach Anspruch 11, wobei die Gerüstregion der variablen Domäne der schweren Kette der Gerüstregion der variablen Domäne der schweren Kette des Proteins NEW homolog ist.
  15. Antikörper nach Anspruch 14, wobei die variable Region der schweren Kette die Aminosäuresequenz, die in der obersten Zeile von Fig. 6 oder 7 gezeigt ist, besitzt.
  16. Antikörper nach einem der Ansprüche 11 bis 15, wobei die Gerüstregion der variablen Domäne der leichten Kette der Gerüstregion der variablen Domäne des Proteins REI homolog ist.
  17. Antikörper nach Anspruch 16, wobei die leichte Kette die Aminosäuresequenz, die in der obersten Zeile der Fig. 3 gezeigt ist, besitzt.
  18. Pharmazeutisches Präparat, umfassend einen pharmazeutisch verträglichen Träger oder ein pharmazeutisch verträgliches Verdünnungsmittel und als Wirkstoff einen Antikörper nach einem der Ansprüche 10 bis 17.
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ATE148172T1 (de) 1997-02-15
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